Sara C. Antunes

Toxicity evaluation of three pesticides on non-target aquatic and soil organisms: commercial formulation versus active ingredient

The Ecological Risk Assessment of pesticides requires data regarding their toxicity to aquatic and terrestrial non-target species. Such requirements concern active ingredient(s), generally not considering the noxious potential of commercial formulations. This work intends to contribute with novel information on the effects of short-term exposures to two herbicides, with different modes of action (Spasor®, Stam Novel Flo 480®), and an insecticide (Lannate®), as well as to corresponding active ingredients (Glyphosate, Propanil and Methomyl, respectively). The microalga Pseudokirchneriella subcapitata (growth inhibition), the cladoceran Daphnia magna (immobilisation), and the earthworm Eisenia andrei (avoidance behaviour) were used as test species. Both herbicides were innocuous to all test organisms at environmentally realistic concentrations, except for Stam and Propanil (highly toxic for Pseudokirchneriella; moderately toxic to Daphnia). Lannate and Methomyl were highly toxic to Daphnia and caused Eisenia to significantly avoid the spiked soil at realistic application rates. The toxicity of formulations either overestimated (e.g. Stam/Propanil for P. subcapitata) or underestimated (e.g. Stam/Propanil for D. magna) that of the active ingredient.

The effect of environmental parameters and cyanobacterial blooms on phytoplankton dynamics of a Portuguese temperate Lake

The increasing occurrence of cyanobacterial blooms in freshwaters is of great concern due to the ability of many cyanobacteria to produce cyanotoxins. In the present work, the eutrophied Vela Lake (Central Portugal), used for recreational purposes and as a water source for agriculture, was monitored every fortnight between 2000 and 2001. Phytoplankton diversity and densities were measured and correlated to environmental parameters. A seasonal phytoplanktonic succession was observed and it was mainly correlated with conductivity, temperature, total suspended solids and nutrients availability (particularly phosphorus). Diatoms were dominant during winter months (inferior temperatures and higher nutrients availability) followed by green algae in early spring and then cyanobacteria from late spring until early autumn (less nutrient availability and higher temperatures). A massive cyanobacterial bloom of Aphanizomenon flos-aquae occurred early in May 2001 and was preceded by the lowest nitrogen levels measured in the water during all the study period. At the time of this bloom senescence, dissolved oxygen was severely depleted and a massive death of ichthyofauna was recorded. A Microcystis aeruginosa bloom was also detected in July 2001 and it occurred following a rapid decrease in abundance of green algae and diatoms. By considering not only the environmental parameters but also the occurrence of cyanobacterial blooms as explanatory variables in a canonical correspondence analysis, the variance explained for the phytoplanktonic assemblage during the study period was increased in about 7% achieving a total of 61.0%, indicating a correlation that may be due to the known competitive advantage and/or allelopathy of the bloom-forming cyanobacteria towards microalgae.

Biochemical effects of acetaminophen in aquatic species: edible clams Venerupis decussata and Venerupis philippinarum

Acetaminophen (paracetamol) is one of the most used pharmaceutical drugs, due to its antipyretic and analgesic properties that turn it into a primary choice in varied pathologies and conditions. However, and despite its massive use, acetaminophen is not exempt of adverse effects, especially when administered in over dosage, which are related to the formation of toxic metabolites by oxidative pathways. It is thus possible to observe that toxicity caused by acetaminophen is usually mediated by reactive oxygen species and can result in multiple effects, ranging from protein denaturation to lipid peroxidation and DNA damage. The occurrence of acetaminophen has been reported in the aquatic environment, being important to address the potential exertion of toxic effects on nontarget environmentally exposed organisms. The present study intended to characterize the effects of acute acetaminophen exposure on physiological traits (antioxidant defense, oxidative damage) of two species of bivalves, namely, the edible clams Venerupis decussata and Venerupis philippinarum. Results showed a significant increase in all oxidative stress biomarkers, evidencing the bioactivation of acetaminophen into a deleterious prooxidant, triggering the onset of deleterious effects. Furthermore, strong interspecific differences were observed among responses of the two tested species, which was a major issue due to intrinsic ecological implications when one considers that both species share the same habitat.

Chronic responses of different clones of Daphnia longispina (field and ephippia) to different food levels

Abstract Resting eggs are a fundamental reproductive strategy among freshwater cladocerans. Under adverse environmental conditions, whole Daphnia populations can disappear from a lake and a new community will arise from ephippial eggs. Since these new populations are subjected to genetic variation, their responses to environmental stress or contaminants can be different from the “original” population. In the present study, life history responses (reproduction and growth) of Daphnia longispina to different food concentrations was studied. Two Daphnia populations were tested: (a) field clones and (b) ephippial clones. Food ( Selenastrum capricornutum ) concentration was the stressor tested (absence of food, and low to high food concentrations). The results showed that reproductive responses of D. longispina to the tested food concentrations varied among clones, independently from their origin.

Short-term effects of neuroactive pharmaceutical drugs on a fish species: Biochemical and behavioural effects

The presence of pharmaceutical residues in the aquatic environment is receiving great attention since significant levels of contamination have been found, not only in sewage treatment plant effluents, but also in open waters. In our study, the toxicity of three anticonvulsant drugs commonly found in the environment (diazepam, carbamazepine, and phenytoin) was evaluated in Lepomis gibbosus (pumpkinseed sunfish). This study focused on oxidative stress parameters, namely: glutathione reductase (GRed), glutathione S-transferases (GSTs), catalase (CAT), and lipid peroxidation (thiobarbituric acid reactive substances, TBARS) in the hepatic, digestive, and gill tissues of exposed animals. Simultaneously, we assessed the effects of these drugs in terms of behavioural parameters, such as scototaxis and activity. Exposure to diazepam caused an increase in GST activities in the gills and an inhibition of GRed in the digestive tract, relative to control, suggesting an antioxidant response. It also caused fish to spend more time swimming and less time in a refuge area (black compartment of an aquarium). Exposure to carbamazepine caused an increase in GSTs and GRed activity in the digestive tract, which is not always consistent with the literature. A significant positive correlation was found between carbamazepine concentration and time spent in motion and a negative correlation with time spent in black compartment. Exposure to phenytoin was responsible for adaptive responses in the activities of CAT and GSTs (in the liver), but it did not elicit any behavioural alterations. Although all three drugs seemed to induce oxidative stress in some organs, peroxidative damage (measured as TBARS concentrations) was not found at the selected range of concentrations. Our results enlighten the need for more research on the ecological consequences of pharmaceuticals in the aquatic environment, especially drugs that interfere with the CNS and behaviour, because the net outcome of these effects may be difficult to predict.

Biological treatment of the effluent from a bleached kraft pulp mill using basidiomycete and zygomycete fungi

Three white-rot fungi (Pleurotus sajor caju, Trametes versicolor and Phanerochaete chrysosporium) and one soft-rot fungi (Rhizopus oryzae) species confirmed their potential for future applications in the biological treatment of effluents derived from the secondary treatment of a bleached kraft pulp mill processing Eucalyptus globulus. Among the four species P. sajor caju and R. oryzae were the most effective in the biodegradation of organic compounds present in the effluent, being responsible for the reduction of relative absorbance (25-46% at 250 nm and 72-74% at 465 nm) and of chemical oxygen demand levels (74 to 81%) after 10 days of incubation. Laccase (Lac), lignin (Lip) and manganese peroxidases (MnP) expression varied among fungal species, where Lac and LiP activities were correlated with the degradation of organic compounds in the effluent treated with P. sajor caju. The first two axes of a principal component analysis explained 88.9% of the total variation among sub-samples treated with the four fungus species, after different incubation periods. All the variables measured contributed positively to the first component except for the MnP enzyme activity which was the only variable contributing negatively to the first component. Absorbances at 465 nm, LiP and Lac enzyme activities were the variables with more weight on the second component. P. sajor caju revealed to be the only species able to perform the biological treatment without promoting an increment in the toxicity of the effluent to the Vibrio fischeri, as it was assessed by the Microtox assay. The opposite was recorded for the treatments with the other three species of fungus. EC(50-5 min) values ranging between 28 and 57% (effluent concentrations) were recorded even after 10 to 13 days of treatment with P. chrysosporium, R. oryzae or with T. versicolor.

Rotifer community structure in three shallow lakes: seasonal fluctuations and explanatory factors

The present work aimed at studying the rotifer communities of three shallow eutrophic lakes in Portugal (lakes Mira, Vela and Linhos). At the time of the study, Mira and Vela faced large inputs of allochthonous nutrients, while Linhos was facing terrestrialisation, with cycles of dominance-senescence of macrophytes. The three lakes differed in terms of their abiotic features, with Linhos presenting very high nutrient levels and low pH, while Vela and Mira shared most of the characteristics. The rotifer communities of these two lakes were poorly diversified but highly abundant (max. > 2000 ind l−1), with a clear dominance of eurytopic euplanktonic species (mainly Keratella cochlearis). On the other hand, Linhos presented lower abundances (<1000 ind l−1) but higher species richness, mainly due to macrophyte-associated taxa, such as the littoral genera Lepadella, Testudinella and Squatinella. In all lakes, summertime represented a peak in terms of abundance and diversity. Canonical correspondence analysis (CCA) identified two main environmental gradients that shape up the rotifer assemblages: a temporal gradient, mainly related to temperature, and a eutrophy gradient, associated with nitrogenous nutrients. The latter gradient is clearly dependent on between-lake variation, due to the high nutrient levels observed in lake Linhos. Variance partitioning using CCA revealed that the largest portion (27.5%) of the total variation explained (52.1%) was attributed to the interaction between lake and environmental variables.

Toxicity and organic content characterization of olive oil mill wastewater undergoing a sequential treatment with fungi and photo-Fenton oxidation.

Olive oil mill wastewater (OOMW) is responsible for serious environmental problems. In this study, the efficiency of two treatments involving fungi and photo-Fenton oxidation, sequentially applied to OOMW was analyzed for organic compounds degradation and toxicity mitigation. The treatment with fungi (especially Pleurotus sajor caju) of diluted OOMW samples promoted a reduction of their acute toxicity to Daphnia longispina. Although this fungi species have not induced significant color reduction it was responsible for 72,91 and 77% reductions in chemical oxygen demand (COD), total phenolic and organic compound contents. After biological treatment, photo-Fenton oxidation seemed to be an interesting solution, especially for color reduction. However, the OOMWs remained highly toxic after photo-Fenton oxidation. Considering the second sequence of treatments, namely photo-Fenton oxidation followed by biological treatment, the former revealed, once more, a great potential because it can be applied to non-diluted OOMW, with significant reductions in COD (53-76%), total phenolic content (81-92%) and organic compounds content (100%). Despite fungal species still have demonstrated a high capacity for bioaccumulation of organic compounds, resulting from photo-Fenton oxidation, the biological treatment did not cause substantial benefits in terms of COD, total phenolic content and toxicity reduction.

Histopathological changes and erythrocytic nuclear abnormalities in Iberian green frogs (Rana perezi Seoane) from a uranium mine pond

In spite of their sensitivity to anthropogenic stressors, adults of Rana perezi Seoane were found inhabiting effluent ponds from a uranium mine. Due to the presence of such organisms in this environment, it becomes of paramount importance to assess the damages induced by local contamination on these aquatic vertebrates, in order to integrate this information on a site-specific risk assessment that is being carried out in the area. To attain this purpose an ethically and statistically acceptable number of green frogs were captured in the mine pond (M) and in a pristine river (VR), a few kilometres from the mine. Bioaccumulation of metals and histopathological alterations were evaluated in the liver, kidneys, spleen, lungs and testes of the animals. Simultaneously, blood samples were collected for the evaluation of genotoxic damage on erythrocytes. Animals captured in the M pond showed significantly increased levels of Be, Al, Mn, Fe and U in the liver, as well as Pb and U in the kidney. The liver was the main target organ for the bioaccumulation of Be, Al, Fe and U. However, renal histopathologies were more severe than those of liver. The main tissue alterations recorded in animals from the mine were: a slight increase in melanomacrophagic centers (MMC) in liver, lung and kidneys; dilatation of the renal tubules lumen associated with tubular necrosis. A significantly higher number of erythrocytic abnormalities (lobed, notched and kidney shaped nuclei and micronuclei) were recorded in frogs from M than in frogs from VR, along with a significantly lower frequency of immature erythrocytes. Both observations suggested that the removal of abnormal blood cells might be compromised.

Bait-lamina assay as a tool to assess the effects of metal contamination in the feeding activity of soil invertebrates within a uranium mine area.

As part of the tier 2 of a site-specific risk assessment, this study was the first reporting an intensive in situ application of the bait-lamina assay; two exposure periods (7 and 14 days) were tested during four seasons in ten different sites, within a uranium mine area and at two different depths. The most contaminated sites (by deposition of sludge from the effluent treatment pond) were discriminated after 14 days of exposure because extremely low percentages of feeding activity were recorded. Previous sub-lethal ecotoxicological assays, already had demonstrated that the habitat function of these soils is compromised. Nevertheless, seasonality has proved to have a significant influence on responses. Thus to strength conclusions about the impact of contaminants, the in situ bait-lamina assay should be performed on different annual seasons, at least for temperate regions. It was also found that some environmental parameters (e.g. soil moisture and litter) can act as confounding factors in the bait-lamina assay.